Pollen anomaly - a clue to natural hybridity in Argemone (Papaveraceae)

Studies in the pollen morphology of Argemone mexicana (a diploid species, 2n=28), A. ochroleuca (a tetraploid, 2n=56), the wild populations of which growing side by side, and of a triploid (2n=42) observed in the population of A. ochroleuca, have been carried out using light and scanning electron microscope. Pollen grains in both the species are 3 (-4)-zonocolpate with reticulate exine surface, and are distinguishable only on the basis of pollen size, the tetraploid species having larger pollen grains. In the triploid plant, a large number of grains deviates from the normal 3-zonocolpate condition showing a number of apertural types, such as 1-aperturate-operculate, 2-colpate, 3-syncolpate, 4-zonocolpate and spiraperturate. Besides, planaperturate 3-colpate-trilobed grains also occur. The triploid is also characterised by 'minigrains' (size 13-19 μm), a feature not observed in A. mexicana and A. ochroleuca. Since these species are reported to be genetically close, the occurrence of the triploids (forming nearly 5% of the population of A. ochroleuca) indicates the possibility of a natural hybridization between A. ochroleuca and A. mexicana, which is well pronounced on its anomalous pollen features serving as marker for such a hybrid occurring in nature.     

Alkaloid chemotaxonomy of Nordic Papaver sect. Scapiflora (Papaveraceae)

A chemotaxonomic study of the Nordic Papaver species belonging to the Scapiflora group has been carried out by means of gas chromatography (GC), mass spectro-graphy (MS), and thin layer chromatography (TLC). The data obtained were analysed by the aid of hard and fuzzy clustering. Of the alkaloids identified, protopine and allocryptopine were found in all species investigated (i.e. Papaver radicaium - including all subspecies - P. dahlianum, P. lapponicum , and P. laesiadianum ). Protopine and allocryptopine were the only alkaloids found in P. dahlianum, P. lapponicum , and P. laesiadianum . In addition O -methylthalisopavine was found in all investigated subspecies of P. radicatum; reframine in P. radicatum sspp. ovatilobum, gjaerevollii, groevudalense, intermedium, oeksendalense, radicatum, relictum , and subglobosum; reframidine in ssp. hyperboreum; cryptopine in sspp. ovatilobum, gjaerevollii, groevudalense, relictum, subglobosum, macrostigma , and hyperboreum; muramine in sspp. macrostigma and hyperboreum; and amurine in sspp. relictum and subglobosum .
These chemotaxonomic results confirm the systematics of the Nordic Papavers of the sect. Scapiflora introduced by Knaben who based the taxonomy on morphological and cytological characteristics. Moreover, the content of alkaloids in the different isolated Nordic Papaver populations may be of plant geographical importance. There are, for instance, a marked chemical difference between all the South Norwegian subspecies of P. radicatum and the North Norwegian P. radicatum ssp. subglobosum on one hand and the rest of the North Norwegian subspecies on the other.     

In vitro adventitious shoot formation from leaf cultures of Clerodendrum inerme (L) Gaertn.

In vitro adventitious shoots (about 28) of Clerodendrum inerme were regenerated from leaf segments on MS medium containing BA (4 mg/L). These shoots developed directly from the leaf explants without callusing after 5 weeks. Leaf explant when cultured in MS medium containing BA (2 mg/L) and NAA (0.5 mg/L) developed compact callus that became nodular and regenerated shoots (about 50) after 5 weeks. The in vitro developed shoots were rooted in MS medium supplemented with IAA (2 mg/L). The hardened plantlets were successfully established in the field with 90% survival.     

Prevalence and effects of Argemone Mexicana (Papaveraceae) on biodiversity in Ethiopia

The prevalence and potentials of Argemone mexicana on biodiversity was assessed at Farta, Gondar and Bahir Dar zuria districts. The study areas were stratified into 100 m by 5 m as roadside, fallow land and farmland transect belts. Its effects as poultry feed component were studied on three poultry breeds. Body weights, abnormalities in appetite, egg production and health were recorded. The results indicated that the mean prevalence of Argemone per transect belt in roadside land (17.41 ± 5.9) was higher than that of fallow land (3.35 ± 0.8) and farmland (2.65 ± 0.8) at Farta district. The number and distribution of native plant species under the shed of A. mexicana in roadside, fallow land and farmland per transect belts were 1.15 ± 1.6, 2.25 ± 1.8 and 1.15 ± 0.5, respectively. The number and distribution of Argemone plants in the roadside land per transect increased by 163.4%, 158.5% and 151.9% from 2006 to 2010 in above respective districts. The mean losses in appetite, body weight and egg production were higher in exotic and cross bred than in indigenous chicken ecotypes. Therefore, A. mexicana had negative impacts on biodiversity, native plant species and when used as animal feed components.     

Vindoline synthesis in in vitro shoot cultures of Catharanthus roseus

Vindoline, the major alkaloid in cultures of Catharanthus roseus shoots, reached 2 mg g(-1) dry wt after 27 d in culture. Maximal vindoline accumulation coincided with maximum activities of deacetoxyvindoline 4-hydroxylase, deacetylvindoline acetyl-CoA acetyl transferase and tryptophan decarboxylase. Shoot exposure to jasmonate shortened the time required for the maximal vindoline accumulation to 14 d.     

Indoleamines and calcium channels influence morphogenesis in in vitro cultures of Mimosa pudica L.

The present article reports the interplay of indoleamine neurohormones viz. serotonin, melatonin and calcium channels on shoot organogenesis in Mimosa pudica L. In vitro grown nodal segments were cultured on MS medium with B5 vitamins containing Serotonin (SER) and Melatonin (MEL) at 100 µM and indoleamine inhibitors viz. serotonin to melatonin conversion inhibitor p-chlorophenylalanine (p-CPA) at 40 µM, serotonin reuptake inhibitor (Prozac) 20 µM. In another set of experiment, calcium at 5 mM, calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) 100 µM, and calcium channel blocker varapamil hydrochloride (1 mM) a calcium chelator EGTA (100 µM) were administered to the culture medium. The percentage of shoot multiplication, endogenous MEL and SER were monitored during shoot organogenesis. At 100 µM SER and MEL treatment 60% and 70% explants responded for shoot multiplication respectively. Medium supplemented with either SER or MEL along with calcium (5 mM) 75%–80% explants responded for organogenesis. SER or MEL along with calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) at 100 µM 70% explants responded for shoot multiplication. p-CPA, prozac, verapamil and EGTA, shoot multiplication was reduced and endogenous pools of SER, MEL decreased by 40–70%. The results clearly demonstrated that indoleamines and calcium channels positively influenced shoot organogenesis in M. pudica L.     

DNA,peroxidase profile,and biochemical composition changes during the treatment in vitro of pollen of Argemone mexicana L. with SO 2

The allergenic pollen of Argemone mexicana L. a common road-side weed was subjected to artificial SO 2 fumigation at a concentration of 100ppm for 24 hours, 48 hours and 72 hours and its effect on the total carbohydrate, lipid, free amino acid, DNA and RNA content as well as peroxidase isozyme and DNA profile studied. In comparison to the control (pollen exposed to charcoal-filtered air), the carbohydrate, lipid, DNA and RNA content decreased with the increase in the time of exposure to SO 2 , where as the percentage composition of free amino acid increased. Contrary, to this, the activity of peroxidase was found to increase with a change in the iso-peroxidase zymogram showing three new anionic isozyme bands. SO 2 affected the total DNA profile leading to the gradual breakdown of DNA with several bands observed on agarose gel electrophoresis with an increase in the time of fumigation.     

Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L.

In contrast to undifferentiated cell suspension cultures of Digitalis lanata, photomixotrophic shoot cultures of Digitalis purpurea accumulate cardiac glycosides in substantial concentrations. They are used to investigate enzymes of the cardenolide pathway. All cardenolides are 5-configurated. The progesterone 5-reductase and the 3-hydroxysteroid-5-oxidoreductase are present in shoot cultures but not in undifferentiated cell cultures. These enzymes provide precursors for cardenolides, whereas the presence of the progesterone 5-reductase, also present in shoot cultures, is discussed with regard to its role in phytosterol biosynthesis and may be attributed to the general steroid pathway. The progesterone 5-reductase had an activity maximum during the early growth period seven days after onset of cultivation, whereas the corresponding progesterone 5-reductase activity was highest on day 11. The maximum cardenolide accumulation was after 24 days. The enzyme activities present in crude extracts from shoot cultures were characterized with regard to their requirements for NADPH and NADH, pH-optimum, temperature optimum, affinity to their substrates and their localization in the cell. The progesterone 5-reductase was purified 769-fold.Abbreviations DW dry weight - FW fresh weight - PVP polyvinylpyrrolidone     

Lenticel hypertrophy in shoot cultures of Ceratonia siliqua L.

Numerous white surface proliferations appeared in cultures of Ceratonia siliqua L. grown three to four weeks on medium containing 0.5 mg l^-1 BA and 0.1 mg l^-1 IBA. It was histologically confirmed that these proliferations were hypertrophied lenticels. Proliferations appeared first at the basal shoot internode and gradually spread acropetally, covering eventually the whole shoot except the uppermost internodes. Increase of BA concentration in the medium increased both the number of hypertrophied lenticels per shoot and the shoot multiplication index.Abbreviations BA 6-benzylamino-purine - IBA indole-3-butyric acid Dubravka Bojovi-Cveti deceased July 8, 1991.     

De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene

Role of ethylene in de novo shoot morphogenesis from explants and plant growth of mustard ( Brassica juncea cv. India Mustard) in vitro was investigated, by culturing explants or plants in the presence of the ethylene inhibitors aminoethoxyvinylglycine (AVG) and AgNO₃. The presence of 20 μ M AgNO₃ or 5 μ M AVG in culture medium containing 5 μ M naphthaleneacetic acid and 10 μ M benzyladenine were equally effective in promoting shoot regeneration from leaf disc and petiole explants. However, AgNO₃ greatly enhanced ethylene production which reached a maximum after 14 days, whereas ethylene levels in the presence of AVG remained low during 3 weeks of culture. The promotive effect of AVG on shoot regeneration was overcome by exogenous application of 25 μ M 2-chloroethylphosphonic acid (CEPA), but AgNO_3-induced regeneration was less affected by CEPA. For whole plant culture, AVG did not affect plant growth, although it decreased ethylene production by 80% and both endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC by 70–80%. In contrast, AgNO₃ stimulated all 3 parameters of ethylene synthesis. Both AgNO₃ and CEPA were inhibitory to plant growth, with more severe inhibition occuring in AgNO₃. Leaf discs derived from plants grown with AVG or AgNO₃ were highly regenerative on shoot regeneration medium without ethylene inhibitor, but the presence of AgNO₃ in the medium was inhibitory to regeneration of those derived from plants grown with AgNO₃.     

Enhanced production of withaferin-A in shoot cultures of Withania somnifera (L) Dunal

Withania somnifera (L) Dunal, commonly known as ashwagandha or Indian ginseng, is the source of large number of pharmacologically active withanolides. Withaferin-A (WS-3), a major withanolide of W. somnifera, has been proven to be an effective anti-cancer molecule. In this study, a liquid culture system for shoot proliferation, biomass accumulation and withaferin-A production of an elite accession (AGB002) of W. somnifera was investigated. The nodal explants cultured on Murashige and Skoog (MS) semi-solid medium supplemented with various concentrations of 6-benzyl adenine (BA) and Kinetin (Kn) elicited varied responses. The highest number of regenerated shoots per ex-plant (35?±?3.25) and the maximum average shoot length (5.0?±?0.25 cm) were recorded on MS medium supplemented with BA (5.0 μM). The shoots were further proliferated in half and full strength MS liquid medium supplemented with the same concentration BA. It was interesting to note that shoots cultured on MS half strength liquid medium fortified with 4 gL-1 FW (fresh weight) shoot inoculum mass derived from 5 week old nodal explants of W. somnifera showed highest accumulation of biomass and withaferin A content in 5 weeks. Withaferin A was produced in relatively high amounts (1.30 % and 1.10 % DW) in shoots cultured in half and full strength MS liquid media respectively as compared to natural field grown plants (0.85 % DW). A considerable amount of the withaferin A was also excreted in the culture medium. Successful proliferation of shoots in liquid medium and the synthesis of withaferin A in vitro opens new avenues for bioreactor scale-up and the large-scale production of the compound.     

Adventitious shoot regeneration in cultures of Humulus lupulus L. (hop) cvs. Brewers Gold and Nugget

 A very efficient protocol for plant regeneration from two commercial Humulus lupulus L. (hop) cultivars, Brewers Gold and Nugget has been established, and the morphogenetic potential of explants cultured on Adams modified medium supplemented with several concentrations of cytokinins and auxins studied. Zeatin at 4.56 μm produced direct caulogenesis and caulogenic calli in both cultivars. Subculture of these calli on Adams modified medium supplemented with benzylaminopurine (4.4 μm) and indolebutyric acid (0.49 μm) promoted shoot regeneration which gradually increased up to the third subculture. Regeneration rates of 60 and 29% were achieved for Nugget and Brewers Gold, respectively. By selection of callus lines, it has been possible to maintain caulogenic potential for 14 months. Regenerated plants were successfully transferred to field conditions. Received: 10 March 1997 / Revision received: 12 November 1997 / Accepted: 22 November 1998     

Glutamine improves shoot morphogenesis in chickpea (Cicer arietinum L.)

The use of glutamine has been shown to increase the frequency of organogenesis and regeneration in the in vitro culture of several plants. The effect of glutamine on hormone-induced multiple shoot formation in desi and kabuli genotypes of chickpea (C-235 and PUSA-1053) were evaluated. Embryo axes with or without attached cotyledons were cultured in thidiazuron (TDZ) or 6-benzylaminopurine (BAP)-containing medium, respectively, with various concentrations of glutamine. Glutamine improved and prolonged the multiple shoot regeneration ability of the embryo axis. Chickpea embryo axis with attached cotyledon and cultured in TDZ-containing medium showed improved and prolonged shoot regeneration with 5 mM glutamine, while embryo axis without cotyledon and cultured in BAP-containing medium showed prolonged regeneration ability in 10 mM glutamine. Glutamine, however, did not serve as a substitute for cotyledon. Desi genotype (C-235) showed better response for multiple shoot formation as compared to the kabuli genotype (PUSA-1053). Glutamine at a concentration of 5 mM also improved root formation in excised in vitro shoots.     

Vindoline formation in shoot cultures of Catharanthus roseus is synchronously activated with morphogenesis through the last biosynthetic step

Background and Aims

The Madagascar periwinkle (Catharanthus roseus) produces the monoterpenoid alkaloid vindoline, which requires a specialized cell organization present only in the aerial tissues. Vindoline content can be affected by photoperiod and this effect seems to be associated with the morphogenetic capacity of branches; this association formed the basis of the study reported here.

Methods

Vindoline-producing in vitro shoot cultures were exposed either to continuous light or a 16-h photoperiod regime. New plantlet formation and alkaloid biosynthesis were analysed throughout a culture cycle.

Key Results

In cultures under the photoperiod, the formation of new plantlets occurred in a more synchronized fashion as compared to those under continuous light. The accumulation of vindoline in cultures under the photoperiod occurred in co-ordination with plantlet formation, in constrast to cultures under continuous light, and coincided with a peak of activity of deacetylvindoline acetyl CoA acetyltransferase (DAT), the enzyme that catalyses the last step in vindoline biosynthesis. When new plantlet formation was blocked in cultures under the photoperiod by treatment with phytoregulators, vindoline synthesis was also reduced via an effect on DAT activity. No association between plantlet formation and other biosynthetic enzymes, such as tryptophan decarboxylase (TDC) and deacetoxyvindoline 4-hydroxylase (D4H), was found. Effects of light treatment on vindoline synthesis were not mediated by ORCA-3 proteins (which are involved in the induction of alkaloid synthesis in response to elicitation), suggesting that the presence of a different set of regulatory proteins.

Conclusions

The data suggest that vindoline biosynthesis is associated with morphogenesis in shoot cultures of C. roseus.Key words: Catharanthus roseus, deacetylvindoline acetyl CoA acetyltransferase, DAT, in vitro shoot cultures, morphogenesis, vindoline     

In vitro propagation of shoot buds of Carica papaya L. (caricaceae) var. Honey Dew

Shoot buds from the saplings and the fruit bearing plants of Carica papaya L.. var. Honey Dew (papaya) initially treated with Gentamycin were cultured in modified MS media, each with a different hormonal combination, for the establishment of cultures and multiplication and rooting of plants. About 43% of explants from fruit bearing plants and 69% of those from saplings remained free of contamination and retained regeneration capacity when treated in 500 mg/l Gentamycin. For the establishment of the explants a medium containing 1 mg/l GA₃ and 2 mg/l kinetin was necessary. When established buds were transferred to medium containing 1 mg/l NAA and 3 mg/l kinetin, calli were initiated at cut ends of shoot buds; multiplication started on transfer to NAA (0.1 mg/l) and BAP (0.5 mg/l) medium. Cultures have been maintained for the last twenty months without any loss in multiplication rate. Rooting was induced in medium with reduced salt concentration containing 2 mg/l IBA. Shoot elongation was induced after prolonged culture in the same rooting medium.Abbreviations MS Murashige and Skoog, 1962 - SH Schenk and Hildebrandt, 1972 - GA₃ Gibberellic acid - Kn Kinetin - NAA Napthaleneacetic acid - BAP 6 -Benzylaminopurine - IBA Indole-3-butyric acid - IAA Indole-3-acetic acid     

Role of polyamines on de novo shoot morphogenesis from cotyledons of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro

Summary The promotive effect of ethylene inhibitors (Els), i.e. AgNO₃ and aminoethoxyvinylglycine (AVG) on de novo shoot regeneration from cultured cotyledonary explants of Brassica campestris ssp. pekinensis cv. Shantung in relation to polyamines (PAs) was investigated. The endogenous levels of free putrescine and spermidine in the explant decreased sharply after 1–3 days of culture, whereas endogenous spermine increased, irrespective of the absence or presence of Els. AgNO₃ at 30 M did not affect endogenous PAs during two weeks of culture. In contrast, explants grown on medium containing 5 M AVG produced higher levels of free putrescine and spermine which increased rapidly after three days and reached a peak at 10 days. An exogenous application of 5 mM putrescine also resulted in a similar surge of endogenous free spermine of the explant. More strikingly, shoot regeneration from explants grown in the presence of 1–20 mM putrescine, 0.1–2.5 mM spermidine, or 0.1–1 mM spermine was enhanced after three weeks of culture. However, exogenous PAs generally did not affect ethylene production, and endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC of the explant. This study shows the PA requirement for shoot regeneration from cotyledons of B. campestris ssp. pekinensis in vitro, and also indicates that the promotive effect of PAs on regeneration may not be due to an inhibition of ethylene biosynthesis.Abbreviations PAs polyamines - AVG aminoethoxyvinylglycine - SAM S-adenosylmethionine - ACC 1-aminocyclopropane-1-carboxylate - Els ethylene inhibitors